1. Field of the Invention
The present invention relates to a quite novel method of measuring physical properties by a super-thin liquid membrane forming mode and a quite novel interface reaction detection type biosensor by a super-thin liquid membrane forming mode applicable regardless of whether it is a solid, liquid or gaseous object to be measured.
2. Description of the Prior Art
The following methods and the like have been known as a method of measuring physical properties using a small quantity of sample:
(1) A flow-through type method as shown in FIG. 20(A). A liquid sample c, as an object to be measured, is introduced at an appointed quantity and followed by another appointed quantity into a capillary b provided with a sample electrode a therein, the electrode being a single-type measuring or reference electrode, or complex-type measuring and reference electrode.
(2) A drop type method as shown in FIG. 20(B). An appointed quantity of liquid sample c, as an object to be measured, is introduced drop by drop by means of a micropipette (not shown) into a sample-introducing concave portion d provided with a sample electrode a in a bottom portion thereof.
(3) A method as shown in FIG. 20(C). An absorption member e impregnated with a liquid sample c, as an object to be measured, is placed on a sample electrode a in a closely contacting relation.
However, all of the above-described various methods (1), (2), (3) are the direct measuring method for the liquid sample c as the object to be measured. Consequently, the measurement is very difficult or impossible according to the items to be measured.
Horiba, Ltd. has developed measuring methods suitable for such cases, that is, indirect measuring methods (4) by a so-called reaction detecting mode or a living body reaction detecting mode, in which the liquid sample c, as the object to be measured, is brought into contact with a solid reactive substance to thereby cause a chemical or physical reaction between them and an electrical change due to the reaction is detected by the sample electrode. Alternatively, the liquid sample c is brought into contact with a membrane fixedly carrying a living body reactive substance acting upon a living body thereon to cause a living body reaction between them and an electrical change due to the reaction is detected by the sample electrode. Not only these measuring methods, but also the biosensor for carrying out the methods have been applied for patent in Japan (see, for example, Japanese patent application No. 3152193/1986).
In addition, the above-described reactive substance or membrane includes, for example, an enzyme-fixed membrane and an antibody-fixed membrane.
According to the indirect measuring method by a reaction detecting mode (4), as shown in FIG. 21, a solid reactive substance g (for example, an enzyme-fixed membrane) is placed on the sample electrode a (for example, a hydrogen peroxide electrode consisting of a platinum electrode and a silver electrode and the like). A buffer solution layer f having an appointed thickness is disposed between the solid reactive substance g and the sample electrode a. An appointed quantity of a liquid sample c, as the object to be measured, is introduced on the solid reactive substance g drop by drop by means of a micropipette h and the like to detect an electrical change due to a chemical or physical reaction by means of the electrode a. The electrical change is produced on an interface between the liquid sample c and the solid reactive substance g, the reaction spreads into the solid reactive substance g, and finally diffuses into the buffer solution layer f, whereby measuring physical properties of the object is accomplished.
In addition, with a biosensor, to which the indirect measuring method (4) by the living body reaction detecting mode is applied, as shown in FIG. 22, a living body reactive membrane g' (for example, an enzyme-fixed membrane) is placed on a sample electrode a (for example, a hydrogen peroxide electrode consisting of a platinum electrode and a silver electrode and the like). A buffer solution layer f having an appointed thickness is disposed between the living body reactive membrane g' and the sample electrode a. An appointed quantity of a sample solution c, as the object to be measured, is introduced on the living body reactive membrane g' drop by drop by means of a micropipette h and the like to detect an electrical change due to a living body reaction by means of the electrode a. The electrical change is produced on an interface between the sample solution c and the living body reactive membrane g'. The reaction then spreads into the living body reactive membrane g', and finally diffuses into the buffer solution layer f, whereby measuring physical properties of the object is accomplished.
However, the measuring methods using a small quantity of sample according to the above-described conventional art and prior art have also shown the following kinds of problems:
(a) An object to be measured must be liquid. Accordingly, in the case where physical properties of solids and gases are to be measured, the solids and gases are first dissolved in an appointed quantity of liquid, such as pure water, which does not have an effect upon the physical properties, to form a liquid sample. That is to say, pretreatment is required, which makes the measurement remarkably troublesome.
(b) Only a small quantity of the liquid sample c, as the object to be measured, is required. But in order to achieve a highly accurate measurement, at least about 0.2 to 0.5 ml of the sample liquid is required for one measurement. In particular, in the case of the above-described indirect measuring method by the reaction detecting mode, a comparatively large amount of liquid sample c is required so that the reaction can be sufficiently spread into the solid reactive substance g. Accordingly, it has been desirable to develop an art capable of achieving a highly accurate measurement using a still smaller amount of sample. In addition, the measurement by means of the electrode a is impossible until the chemical or physical reaction produced on an interface between the liquid sample c, as the object to be measured, and the solid reactive substance g spreads into the solid reactive substance g itself, diffuses into the buffer solution layer f and reaches the electrode a. In short, the reaction is indirectly detected through the reactive substance g itself, which is solid, and the buffer solution layer f for transmitting the reaction. Thus, the reaction-diffusing time is remarkably increased and a sufficiently large stationary detection signal is remarkably difficult to obtain and further, the thickness of the buffer solution layer f is remarkably difficult to control.
In addition, the biosensors according to the above-described prior art have exhibited the problems similar to those in the above-described (b).